Induction of superovulation in cyclic heifers: The inhibitory effect of large doses of PMSG

In two different experiments, superovulation was attempted with a PMSG-PG treatment; a bovine anti-PMSG serum was injected at estrus. After 2500, 5000 and 7500 IU of PMSG injected during the luteal phase, the mean ovulation rates were respectively 16.2 +/- 7.7, 3.2 +/- 2.1, and 1.4 +/- 0.6 in the first experiment (17 heifers) and 18.3 +/- 12.6, 8.5 +/- 8.2, and 2.2 +/- 2.3 in the second (19 heifers). The estradiol-17beta and progesterone patterns and the observations of the ovaries on the day of estrus (Day 0) by ultrasonic echography and on Day 8 by endoscopy show that the ovaries were highly stimulated and suggest that the inhibition observed with the largest doses reflects the absence of the preovulatory LH discharge or its effect.     

Recovery rate and quality of embryos collected from suckled cows and beef heifers after superovulation with PMSG

Recovery rate and embryo quality were investigated in beef heifers and suckled cows following superovulation induced by 2000 IU pregnant mare serum (PMSG) combined with different methods of estrus cycle synchronization (Norgestomet, Prid, Dinolytic, Norgestomet combined with Dinolytic). Genital tracts were flushed upon slaughter with Dulbecco's medium 6.5 to 7.5 days after insemination. Of the heifers, 42 out of 43 responded to treatment. The mean embryo recovery rate, based on the number of corpora lutea, was only 14.8%. Of the 83 embryos recovered, 54.2% had developed to the expected stage and only 40% appeared normal. Of the adult cows, 55 out 58 responded with an embryo recovery rate of 39.5%. Of the 149 embryos recovered, 48.9% had developed to the expected stage and 67.1% of these appeared normal. In both heifers and adult cows, the different methods of estrus synchronization produced no significant differences in recovery rate or embryo quality.     

Factors affecting reproductive performance of Holstein heifers

The objectives were to evaluate factors affecting reproductive performance of dairy heifers. Holstein heifers (6389) were housed in a feed lot located in Parma, ID. Each week heifers weighing > or =290 kg were initiated in the reproductive program, which consisted of one injection of PGF(2alpha) and AI on detection of estrus. Heifers not inseminated by 11 days after the initiation of the breeding program received a second injection of PGF(2alpha). Pregnancy was diagnosed at 40+/-3 and 90+/-3 days after AI. Average daily minimum temperature (ADMnT), average daily maximum temperature (ADMxT), and average daily rainfall (ARF) were recorded between 15 days prior to and 15 days after the day of AI or the day of initiation of the breeding program. Exposure to air temperature was classified as: cold stress (CS=ADMnT< or =4 degrees C), no stress (NS=ADMnT>4 degrees C and ADMxT<29 degrees C), and heat stress (HS=ADMxT> or =29 degrees C). Exposure to rainfall was classified as above (HRF) or below (LRF) the mean for the period in question. Heifers were classified according to body weight at initiation of the breeding program as thin (TH<340 kg); moderate (MD=340-365 kg); and heavy (HY>365 kg). Service sire was associated with conception rate at 40 and 90 days after first AI. Although exposure to air temperature was not correlated with conception rate at 40 days after first AI, heifers exposed to cold stress had smaller conception rates at 90 days after first AI because they were more likely to lose pregnancy between 40 and 90 days of gestation. The proportion of heifers inseminated after initiation of the breeding program was correlated with body weight and exposure to cold stress. Exposure to cold stress was also correlated with the proportion of heifers conceiving within 11 and 22 days after initiation of the breeding program. From this study a correlation was established between body weight and rate of insemination and between the exposure to cold stress and reproductive efficiency of Holstein heifers.     

Preovulatory plasma estradiol-17beta concentrations and ovulation rates in PMSG/anti-PMSG treated heifers

Possibilities for early characterization of the superovulatory response were studied in 41 PMSG/PG-treated dairy heifers, of which 21 received an additional treatment of PMSG-antiserum. Plasma was obtained at 33, 36, 41, 47 and 51 h after PG for hormone analyses. After slaughter at 6 or 7 d after insemination, the number of follicles and corpora lutea (CL) were recorded, and ova were recovered for morphological evaluation. Significant correlations were demonstrated between plasma concentrations of estradiol-17beta (E2) at 33, 36 and 41 h after PG and the ovulation rate (number of CL). Each of these correlations was equal to the one found by using the peak concentration of E2 achieved during the preovulatory E2 surge. In heifers with preovulatory E2 surges, as determined with the blood sampling scheme used, both the ovarian response (number of CL and follicles) and the quality of ova recovered (number of transferable embryos) was clearly better compared to heifers without this surge. None of the parameters studied was affected significantly by treatment with PMSG-antiserum. It is concluded that plasma E2 determinations at fixed times in relation to prostaglandin treatment can be used to characterize the superovulatory response in donor cattle in terms of the ovulation rate and the quality of ova recovered. No evidence was found in favor of using PMSG-antiserum for improving either the superovulatory response or such characterization.     

Factors affecting postpartum ovarian activity in crossbred primiparous tropical heifers

The relationship between postpartum ovarian activity and a total of 9 variables was studied in a dry tropical environment. Primiparous cows (n=61) that had shown no peripartum abnormalities, and were not suckled but milked twice daily, were used in the study. Independent variables included crossbreeding, sex of the calf, season, body condition, weight of cow at calving, age of dam at calving, uterine involution, calf weight and accumulated milk yield. Diet was a controlled variable. Dependent variables were first estrus postpartum and/or first milk progesterone elevation prior to first estrus. A bull fitted with a chin ball marker was used to detect first estrus postpartum, while ovarian structures were palpated per rectum once a week. Progesterone was measured by RIA in milk samples collected twice weekly. First postpartum estrus was detected at 56 +/- 32 days postpartum, a first milk progesterone elevation was observed in 50.8% of cows at 42 +/- 27 days. Cows calving in the dry season had longer intervals and those who calved males had shorter postpartum intervals. Accumulated milk yield affected both intervals significatly (p < 0.01). Weight, age and uterine involution were asociated with first milk progesterone elevation, while crossbreeding, weight at calving and weight postpartum change were associated with the dependent variables.     

Endocrine responses of goats after induction of superovulation with PMSG and FSH

Goats in Group A were pretreated for 9 days with a synthetic progestagen, administered via intravaginal sponge, and 1000 i.u. PMSG s.c. on Day 12 of the oestrous cycle. Goats in Group B had the same PMSG treatment, but not the progestagen pretreatment. Group C goats received a s.c. twice daily injection of a porcine FSH preparation (8 mg on Day 12, 4 mg Day 13, 2 mg Day 14 and 1 mg Day 15). Oestrus was synchronized in all animals by 50 micrograms cloprostenol, 2 days after the start of gonadotrophin treatment. The vaginal progestagen sponges were removed from Group A at the same time. Mean ovulation rate was slightly higher in FSH-treated than in the PMSG-treated animals, whereas the incidence of large follicles that failed to ovulate was significantly elevated in PMSG-treated animals in Group B. More goats in Groups A and B than in Group C exhibited premature luteal failure. Progestagen pretreatment appeared to suppress both follicular and luteal activity, as indicated by numbers of large non-ovulating follicles and by the magnitude and duration of elevated plasma oestradiol levels following PMSG stimulation, and by decreased plasma progesterone levels before and after PMSG treatment. Oestrogenic response to FSH was considerably less than that to PMSG, as indicated both by a considerably shorter duration of elevation of circulating oestradiol levels during the peri-ovulatory period, and by lower maximal oestradiol levels. Differences in the ovarian responses to PMSG and FSH may be attributed primarily to differences in the biological half-life of each preparation.     

Influence of dose, repeated treatment and batch of hormone on ovarian response in heifers treated with PMSG.

The effect of two dose levels (1000 and 2000 i.u.) of three different commercially available batches of PMSG on the ovarian response (ovulations and follicles greater than 10 mn) of 42 heifers was examined in a randomized incomplete block experiment. Each animal was subjected to two consecutive but different treatments. A significant effect of dose was observed and there were fewer ovulations, but no reduction in the number of follicles, after the second PMSG treatment. There was no evidence that the ovarian response was affected by the PMSG batch used.     

Follicular dynamics prior to and during superovulation in heifers

The present ultrasonographic study examined the relationship between certain follicular parameters and the superovulatory response in gonadotropin-stimulated heifers. Thirty heifers received a total of 35 mg FSH twice daily for 4 d and 0.75 mg cloprostenol were given to induce luteolysis and estrus at 72 h after the initial FSH injection. Transrectal ultrasonography was performed once daily from 1 or 2 d before the initial FSH injection and until the day of estrus. The number of small (2 to 4 mm), medium (5 to 9 mm), and large (>/=10 mm) size follicles as well as the diameter of the large follicles were recorded. Embryos were recovered non-surgically 6 or 7 d after estrus, and the number of corpora lutea was determined by palpation per rectum. Heifers with >2 or 0.05). The number of large follicles and the sum of medium and large follicles were positively correlated (r=0.43 and r=0.54, respectively; P<0.05) with the number of corpora lutea palpated on the day of embryo recovery (6 to 7 d after estrus). In conclusion, there was an effect of the day relative to initiation of FSH treatment on all follicular categories in heifers responding positively to superovulation, and there was no effect of side (left or right ovary) or of corpus luteum diameter (ipsilateral or contralateral).     

Genetic and hormonal factors affecting superovulation

Prolific sheep breeds provide the best example of genetic effects on ovarian function that are relevant to the problems of superovulation of domestic livestock. Some of these animals superovulate without any treatment. Their ovaries have been deregulated from the feed-back control systems that normally restrict ovulation rate of sheep and cattle to 1 or 2. The study of prolific sheep highlights the roles of ovarian follicle number, follicle morphology, follicle recruitment, plasma FSH concentrations, inhibin and follicle growth inhibitor in achieving high ovulation rates. These are discussed in relation to the problems of superovulation.Prolific sheep are generally more sensitive to PMSG than other breeds but this may not apply to exogenous FSH. Prolific cattle genotypes, although at an early stage of development, could provide useful guidelines for superovulation in the future. Preliminary data show that they are more sensitive to the ovulatory effect of PMSG and FSH than unselected cattle. Sheep and cattle actively immunized against inhibin or ovarian steriods may be more responsive to treatments designed to induce superovulation. Recent advances in the purification of inhibin and realization of the significance of a locally acting follicle growth inhibitor should result in new developments in this field.     

Endocrinological evaluation of the induction of superovulation with PMSG in water buffalo (Bubalus bubalis)

Ten nonlactating buffalo were superovulated with 3000 IU PMSG. Luteolysis was induced with 500 mug Cloprostenol (PG) 60 and 72 h after PMSG. Five buffalo were alloted for natural mating and five were bred by artificial insemination 60 and 84 h after the first PG treatment. Since four buffalo developed pyometra, only 6 of 10 underwent embryo collection successfully 180 to 190 h after PG. Three buffalo yielded only one morula each, while the remaining three yielded a total of two, three and four morulae and/or blastocysts as well als zero, one and three unfertilized ova, respectively. Six of the ten buffalo were assigned to an intensive blood collection regimen. Mean concentrations of progesterone (ng/ml) increased from 1.9 at PMSG stimulation to 4.8 at induction of luteolysis and decreased to a nadir of 0.2 about 72 h after PG treatment. The preovulatory surge of LH occurred 36 +/- 9 h after PG and was low in magnitude (7.3 +/- 1.3 ng/ml). Stimulation of 3 to 12 follicles resulted in concentrations of estradiol-17beta exceeding 5 pg/ml within 48 h after PMSG treatment and reaching a maximum of 32 +/- 11 pg/ml about the time of the preovulatory surge. Only in two individuals did concentrations decrease below 5 pg/ml within the following 12 h. In the other four buffalo 3 to 10 unovulated structures remained palpable, secreting estradiol-17beta far exceeding the preovulatory concentrations. The fast appearing, low magnitude LH surges were key problems resulting from PMSG treatment. They caused unovulated endocrinologically active follicles. High estrogen levels during the early luteal period may activate subclinical uterine infections, which in turn may negatively affect embryonic development.     

Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation

The concentrations of six steroids and of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in follicular fluid from preovulatory and large atretic follicles of normal Holstein heifers and from preovulatory follicles of heifers treated with a hormonal regimen that induces superovulation. Follicular fluid from preovulatory follicles of normal animals obtained prior to the LH surge contained extremely high concentrations of estradiol (1.1 +/- 0.06 micrograms/ml), with estrone concentrations about 20-fold less. Androstenedione was the predominant aromatizable androgen (278 +/- 44 ng/ml; testosterone = 150 +/- 39 ng/ml). Pregnenolone (40 +/- 3 ng/ml) was consistently higher than progesterone (25 +/- 3 ng/ml). In fluid obtained at 15 and 24 h after the onset of estrus, estradiol concentrations had declined 6- and 12-fold, respectively; androgen concentrations had decreased 10- to 20-fold; and progesterone concentrations were increased, whereas pregnenolone concentrations had declined. Concentrations of LH and FSH in these follicles were similar to plasma levels of these hormones before and after the gonadotropin surges. The most striking difference between mean steroid levels in large atretic follicles (greater than 1 cm in diameter) and preovulatory follicles obtained before the LH surge was that estradiol concentrations were about 150 times lower in atretic follicles. Atretic follicles also had much lower concentrations of LH and slightly lower concentrations of FSH than preovulatory follicles. Hormone concentrations in follicles obtained at 12 h after the onset of estrus from heifers primed for superovulation were similar to those observed in normal preovulatory follicles at estrus + 15 h, except that estrogen concentrations were about 6-40 times lower and there was more variability among animals for both steroid and gonadotropin concentrations. Variability in the concentrations of reproductive hormones in fluid from heifers primed for superovulation suggests that the variations in numbers of normal embryos obtained with this treatment may be due, at least in part, to abnormal follicular steroidogenesis.     

Changes in peripheral inhibin levels and follicular development following treatment of buffalo with PMSG and Neutra-PMSG for superovulation

Fourteen buffalo were synchronized by administration of a prostaglandin (PG) salt Lutalyse in a double injection schedule, with a single intramuscular (im) injection of 25 mg at Day -13, followed by 30 mg and 20 mg im 12 h apart on Day 0 of the experiment. The 30-mg PG injection was designated as 0 h of the experiment. Group I animals (n = 4) received saline and served as the controls, while animals in Groups II and III (n = 5 each) received PMSG (2500 IU im at -48 h. Group III animals were administered 5 ml Neutra-PMSG intravenously at 60 h. Blood samples were collected every 48 h from Day -12 to Day -4, every 24 h from Day -4 to Day 0, every 3 h from Day 1 to Day 4 and every 24 h from Day 5 to Day 10 of experiment for the measurement of peripheral plasma inhibin concentrations by RIA. The number of large follicles (> 10 mm diameter) in animals of Groups II and III was assessed by ultrasonography on Days -2, -1, 0, 1, 2, 5 and 7 of the experiment. Treatment with PMSG of Group II animals resulted in a significant increase (P < 0.05) in plasma inhibin concentrations over that of control animals of Group I at 24 to 99 h, with a peak inhibin concentration of 1.01 +/- 0.31 ng/ml at 48 h. Treatment with Neutra-PMSG in Group III animals caused a significant reduction (P < 0.05) in the peripheral inhibin concentrations at 84 to 120 h and in the number of large unovulated follicles at 168 h compared with that in Group II animals. Peripheral inhibin levels in Group III animals came down to those of Group I after 21 h of Neutra-PMSG treatment. These results suggest that treatment of buffalo with PMSG for superovulation causes a marked rise in peripheral inhibin concentrations. Administration of Neutra-PMSG after PG treatment reduces the peripheral inhibin concentrations and the number of large unovulated follicles.